Physicians treating tuberculosis (TB) today are in desperate need of new, efficient, and effective antibiotics to improve both time of treatment and durability of cure in multi-drug resistant (MDR) as well as uncomplicated TB. In the completed Phase 1 grant (Final Report below), we evaluated an exciting new drug candidate, SQ641. SQ641 has activity superior to Isoniazid (INH) and all other TB drugs and, under appropriate conditions, rapidly reduces Mycobacteria in our two best TB disease models;macrophages and mice. We briefly describe the drug properties, including its exceptional bactericidal activity, its target and mechanism of action (Translocase I), and our efforts to date to develop an oral formulation. In this Phase 2 proposal, we outline the next steps to identify a commercially viable oral formulation(s) of SQ641 for oral treatment regimens, and a parenteral formulation(s) which would have the potential to create a new treatment paradigm;a single, parenteral dose administered at the time of diagnosis. Compared to the current standard of care for TB, both formulations, and associated treatment regimens would dramatically and quickly reduce bacteria in tissues and drastically shorten and improve TB treatment time. INH is used for the first 2 mo of intensive chemotherapy as a cornerstone drug of TB therapy because of its ability to quickly kill rapidly replicating Mycobacterium tuberculosis (MTB) in infected tissues and reduce the number of infectious bacteria in pulmonary secretions. As evidenced by Early Bactericidal Activity (EBA) clinical studies, INH is clearly the most potent of all currently available TB drugs. Interestingly, compared to SQ641, INH is a relatively slow-acting drug in vitro that requires several days to kill MTB, and has a post antibiotic effect (PAE) of only 17 hr. Based on a relatively short half- life, the drug is administered daily. Despite daily administration, recent studies demonstrate that the number of viable bacteria remains sufficient to promote physiologic (Siddiqi et al. 2007) and genotypic drug-resistant bacteria. After 48 hr of exposure to INH, ~10% of MTB are killed, but still morphologically intact. In contrast, SQ641 kills 90% of MTB in this timeframe and the bacteria are lysed (liquid cultures are clear). Moreover, SQ641 has a PAE of 55 hr, 3 times the INH PAE. The best way to prevent drug resistance is to act quickly, before bacteria have a chance to adjust to the presence of the drug. SQ641 is highly effective in preventing development of drug resistant mutants in vitro and has marked synergistic activity in vitro with several antituberculars: Ethambutol (EMB), streptomycin, and the new Sequella drug, SQ109. SQ641 has excellent in vitro activity against drug susceptible and MDR MTB, M. kansasii, M. abscessus, and M. avium, and could also be developed for nontuberculous mycobacterial (NTM) diseases. In spite of the remarkable antibiotic properties of SQ641 against Mycobacteria, a practical method to deliver this drug remains a challenge. We discovered that SQ641 activates the P-glycoprotein (P-gp) drug efflux pump in macrophages, and therefore does not accumulate to bactericidal concentrations inside these important cells that harbor MTB in infected mammals. As expected, P-gp efflux inhibitors markedly enhanced the activity of SQ641 against intracellular MTB;SQ641 was equivalent to or better than INH (the most potent single drug in this test system). In mice, orally administered SQ641 is poorly absorbed. For numerous reasons (patient acceptability, convenience, and cost), oral delivery is the preferred method to administer TB therapy. Poor bioavailability combined with rapid efflux from macrophages meant thinking creatively about drug delivery. Fortunately, we solved both efflux and insolubility problems by combining SQ641 with TPGS, a vitamin E analogue routinely used to improve drug solubility and hence bioavailability (EASTMAN and Company 2005). Stable, 4-6?m (~ the size of Mycobacteria) SQ641-containing TPGS particles were constructed. These particles were easily phagocytosed by macrophages and SQ641 was released inside phagosomes to efficiently and effectively kill intracellular MTB. In both macrophages and mouse models of TB, SQ641/TPGS formulation was as effective as INH. In this Phase 2 proposal, we will evaluate various methods (chemical modification to enhance solubility, alter drug-P-gp interactions, and develop appropriate commercial delivery systems) to solubilize SQ641 and optimize its delivery in vivo. We will explore formulations to determine whether this potent antimicrobial can be delivered orally (combined with current standard-of-care TB drugs or as a replacement for one or more of these drugs) and/or should be delivered parenterally to initiate TB chemotherapy and provide early and prolonged bacterial clearance during the intensive phase of TB treatment.